Chronic rheumatoid arthritis therapy containing IL-6 antagonist as effective component

ABSTRACT

There is provided a synovial cell growth inhibitor, or a pharmaceutical composition for treatment of chronic rheumatoid arthritis based on the synovial cell growth inhibitor.  
     The pharmaceutical composition for treatment of chronic rheumatoid arthritis or synovial cell growth inhibitor contains an IL-6 antagonist, such as IL-6 antibody or IL-6R antibody, as an effective component.

TECHNICAL FIELD

[0001] The present invention relates to a chronic rheumatoid arthritistherapy or synovial cell growth inhibitor comprising an interleukin-6antagonist as an effective component.

BACKGROUND ART

[0002] Chronic rheumatoid arthritis is a systemic chronic inflammatorydisease in which abnormal growth of connective tissue, includingsynovial tissue, occurs in the joints (Melnyk et al., Arthritis Rheum.33: 493-500, 1990). The joints of chronic rheumatoid arthritis patientshave been shown to have marked growth of synovial cells, formation of amultilayer structure due to abnormal growth of the synovial cells(pannus formation), invasion of the synovial cells into cartilage tissueand bone tissue, vascularization toward the synovial tissue, andinfiltration of inflammatory cells such as lymphocytes and macrophages.Mechanisms of onset of chronic rheumatoid arthritis have been reportedto be based on such factors as heredity, bacterial infection and thecontribution of various cytokines and growth factors, but the overallmechanism of onset has remained unclear.

[0003] In recent years, cytokines and growth factors includinginterleukin-1 (IL-1), interleukin-8 (IL-8), tumor necrosis factor α(TNFα), transforming growth factor β (TGFβ), fibroblast growth factor(FGF) and platelet-derived growth factor (PDGF) have been detected inthe synovial membrane and synovial fluid of chronic rheumatoid arthritispatients (Nouri et al., Clin. Exp. Immunol. 55:295-302, 1984; Thorntonet al., Clin. Exp. Immunol. 86:79-86, 1991; Saxne, et al., ArthritisRheum. 31:1041-1045, 1988; Seitz et al., J. Clin. Invest. 87:463-469,1991; Lafyatis et al., J. Immunol. 143:1142-1148, 1989; Melnyk et al.,Arthritis Rheum. 33:493-500, 1990).

[0004] It is believed that IL-1, TNFα and PDGF are particularly powerfulsynovial cell growth factors (Thornton et al., Clin. Exp. Immunol.86:79-86, 1991; Lafyatis et al., J. Immunol. 143:1142-1148, 1989; Gitteret al., Immunology 66:196-200, 1989). It has also been suggested thatstimulation by IL-1 and TNF results in production of interleukin-6(IL-6) by synovial cells (Ito et al., Arthritis Rheum. 35:1197-1201,1992).

[0005] IL-6 is a cytokine also known as B cell-stimulating factor 2 orinterferon β2. IL-6 was discovered as a differentiation factorcontributing to activation of B lymphoid cells (Hirano, T. et al.,Nature 324, 73-76, 1986), and was later found to be a multifunctioncytokine which influences the functioning of a variety of different celltypes (Akira, S. et al., Adv. in Immunology 54, 1-78, 1993). Twofunctionally different membrane molecules are necessary for theinduction of IL-6 activities. One of those is IL-6 receptor (IL-6R), anapproximately 80 KD molecular weight, which binds specifically to IL-6.

[0006] IL-6R exists in a membrane-binding form which is expressed on thecell membrane and penetrates the cell membrane, as well as in the formof soluble IL-6R (sIL-6R) which consists mainly of the extracellulardomain. Another protein is gp130 with a molecular weight ofapproximately 130 KD, which is non-ligand-binding but rather functionsto mediate signal transduction. IL-6 and IL-6R form the complexIL-6/IL-6R which in turn binds with another membrane protein gp130, toinduce the biological activity of IL-6 to the cell (Taga et al., J. Exp.Med. 196:967, 1987).

[0007] It has been reported that the serum or synovial fluid of chronicrheumatoid arthritis patients contains therapies, but since theircontinuous use induces undesirable side effects such as skin tissuedamage and inhibition of adrenal cortex function, drugs with less sideeffects have been sought.

[0008] It is an object of the present invention to provide a novelchronic rheumatoid arthritis therapy without the disadvantages mentionedabove. More specifically, the present invention provides apharmaceutical composition for inhibiting abnormal growth of synovialcells in chronic rheumatoid arthritis, whose effective component is aninterleukin-6 antagonist, as well as a pharmaceutical composition fortreatment of a chronic rheumatoid arthritis having the same effect.

[0009] The present inventors have conducted diligent research on therole of IL-6 on synovial cells from rheumatoid arthritis, during whichno growth of chronic rheumatoid arthritis synovial cells was found withIL-6 alone and a factor other than IL-6 was therefore investigated, andthis has resulted in completion of the present invention based on thediscovery that while IL-6 alone exhibits almost no growth effect onsynovial cells, a powerful synovial cell growth effect occurs in thepresence of both IL-6 and soluble IL-6R, and further that this synovialcell growth effect is suppressed by addition of an antagonist whichinhibits IL-6 activity, such as IL-6 antibody or IL-6R antibody.

[0010] In other words, the present invention relates to a pharmaceuticalcomposition for treatment of a chronic rheumatoid arthritis comprisingan IL-6 antagonist as the effective component. More specifically, thepresent invention relates to a pharmaceutical composition for treatmentof a chronic rheumatoid arthritis comprising an IL-6 antagonist as theeffective component and suppressing abnormal growth of synovial cells.The present invention also relates to a synovial cell growth inhibitorwhose effective component is an IL-6 antagonist. excessive amounts ofinterleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) (Houssiau etal., Arthritis Rheum. 31:784-788, 1988; Hirano et al., Eur. J. Immunol.18:1797-1801, 1988; Yoshioka et al., Japn. J. Rheumatol. in press), andsince similar results have also been obtained in rheumatoid arthritisanimal models (Takai et al., Arthritis Rheum. 32:594-600, 1989; Leistenet al. Clin. Immunol. Immunopathol. 56: 108-115, 1990), it has beensuggested that IL-6 is somehow involved in chronic rheumatoid arthritis.

[0011] However, Japanese Unexamined Patent Publication No. 4-89433discloses that peptides which strongly promote IL-6 production areeffective as therapies for chronic rheumatoid arthritis.

[0012] Also, Higaki et al. have suggested that synovial cells fromchronic rheumatoid arthritis patients have a low growth reaction againstIL-6, and that IL-6 thus has an inhibitory function against growth ofsynovial cells (Clinical Immunology, 22:880-887, 1990). Thus,conflicting reports exist regarding the relationship between IL-6 andchronic rheumatoid arthritis, and the relationship is as yet unclear.

[0013] Recently, Wendling et al. have reported that administration ofanti-IL-6 antibodies to chronic rheumatoid arthritis patientstemporarily alleviates the clinical and biological symptoms, while alsoincreasing IL-6 levels in the serum (J. Rheumatol. 20:259-262, 1993).

[0014] These reports provide no data at all about whether IL-6accelerates growth of chronic rheumatoid arthritis synovial cells or hasan inhibitory effect, and thus it is still unknown whether or not IL-6has a direct effect on synovial cells of chronic rheumatoid arthritispatients.

DISCLOSURE OF THE INVENTION

[0015] Anti-inflammatory steroidal agents such as corticosteroids havebeen used as rheumatoid arthritis

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a graph showing ³H-thymidine uptake into synovial cellsin the presence of either IL-6 or sIL-6R alone and in the presence ofboth IL-6 and sIL-6R.

[0017]FIG. 2 is a graph showing the effect of IL-6 antibody or IL-6Rantibody on ³H-thymidine uptake into synovial cells in the presence ofboth IL-1β and sIL-6R.

[0018]FIG. 3 is a graph showing the effect of IL-6 antibody or IL-6Rantibody on ³H-thymidine uptake into synovial cells in the presence ofboth IL-6 and sIL-6R.

[0019]FIG. 4 is a graph showing the suppressive effect of IL-6R antibodyon the onset of mouse collagen-induced arthritis models.

[0020]FIG. 5 is a graph showing serum anti-collagen antibody levels inarthritic mice.

[0021]FIG. 6 is a photograph of histopathological examination of hindpaw joint of a collagen-arthritis mouse. (a) is a photograph from amouse in an IL-6 receptor antibody-administered group, and (b) is from amouse in a control antibody-administered group. In the IL-6 receptorantibody-administered group, invasion of granulation tissue into thecartilage and bone (chronic proliferative synovitis) was clearlysuppressed.

DETAILED DESCRIPTION OF THE INVENTION

[0022] A pharmaceutical composition for treatment of a chronicrheumatoid arthritis according to the invention is a drug which whenadministered to chronic rheumatoid arthritis patients suppresses growthof synovial cells in joints and has an alleviating and therapeuticeffect on the symptoms.

[0023] The IL-6 antagonist used according to the invention may bederived from any source so long as it is a substance which blocks IL-6signal transfer and inhibits IL-6 biological activity. IL-6 antagonistsinclude IL-6 antibody, IL-6R antibody, gp130 antibody, modified IL-6,antisense IL-6R and partial peptides of IL-6 or IL-6R.

[0024] An antibody used as an antagonist according to the invention,such as IL-6 antibody, IL-6R antibody or gp130 antibody, may be of anyderivation or type (monoclonal, polyclonal), but monoclonal antibodiesderived from mammalian animals are especially preferred. Theseantibodies bind to IL-6, IL-6R or gp130 to inhibit binding between IL-6and IL-6R or IL-6R and gp130 and thus block IL-6 signal transduction,inhibiting IL-6 biological activity.

[0025] The animal species for the monoclonal antibody-producing cells isnot particularly limited so long as it is a mammal, and human antibodiesor antibodies derived from a mammal other than human may be used.Monoclonal antibodies derived from a mammal other than human arepreferably monoclonal antibodies derived from rabbits or rodents becausethey are easier to prepare. There is no particular restriction on therodents, but preferred examples are mice, rats and hamsters.

[0026] Examples of such antibodies which are IL-6 antibodies includeMH166 (Matsuda et al., Eur. J. Immunol. 18:951-956, 1988) and SK2antibody (Sato et al., Journal for the 21st General Meeting of the JapanImmunology Association, 21:116, 1991). Examples of IL-6R antibodiesinclude PM-1 antibody (Hirata et al., J. Immunol. 143:2900-2906, 1989),AUK12-20 antibody, AUK64-7 antibody and AUK146-15 antibody (Intl.Unexamined Patent Application No. WO92-19759). An example of gp130antibody is AM64 antibody (Japanese Unexamined Patent Publication No.3-219894).

[0027] Among these, PM-1 antibody is preferred.

[0028] Monoclonal antibodies may be prepared in the following mannerwhich is based on a known technique. That is, IL-6, IL-6R or gp130 isused as the sensitizing antigen for immunization according to aconventional immunizing method, and the resulting immunocytes are thenfused with known parent cells by a conventional cell fusion method andmonoclonal antibody-producing cells are screened by a conventionalscreening method to prepare the antibodies.

[0029] More specifically, the monoclonal antibodies may be prepared inthe following manner. For example, if the sensitizing antigen is humanIL-6, the antibodies are obtained using the gene sequence for human IL-6disclosed by Hirano et al., Nature, 324:73, 1986. The human IL-6 genesequence is inserted into a publicly expression vector system and usedto transform suitable host cells, after which the desired IL-6 proteinis purified from the host cells or from the culture supernatant and thepurified IL-6 protein is then used as the sensitizing antigen.

[0030] In the case of human IL-6R, the IL-6R protein may be obtained bythe same method as for human IL-6 described above, using the genesequence disclosed in European Patent Application No. EP325474. Twotypes of IL-6R exist, one expressed on the cell membrane and a solubleform (sIL-6R) which is separated from the cell membrane. sIL-6R consistsmainly of the extracellular domain of IL-6R which is attached to thecell membrane, and it differs from the membrane-bound IL-6R in that itlacks the transmembrane domain or the transmembrane domain and theintracellular domain.

[0031] In the case of human gp130, the gp130 protein may be obtained bythe same method as for human IL-6 described above, using the genesequence disclosed in European Patent Application No. EP411946.

[0032] The mammalian animals immunized with the sensitizing antigen arenot particularly restricted, but they are preferably selected inconsideration of their compatibility with the parent cells used for thecell fusion, and generally mice, rats, hamsters and rabbits may be used.

[0033] The immunization of the animals with the sensitizing antigen maybe accomplished by a publicly known method. For example, a conventionalmethod involves intraperitoneal or subcutaneous injection of themammalian animals with the sensitizing antigen. Specifically, thesensitizing antigen is preferably diluted with an equivalent of PBS(Phosphate-Buffered Saline) or physiological saline, suspended and usedtogether with a suitable amount of a conventional adjuvant such asFreund's complete adjuvant if desired, and then administered to themammalian animals a few times every 4-21 days. An appropriate carriermay also be used for immunization with the sensitizing antigen.

[0034] After this immunization and confirmation of increased serumlevels of the desired antibody, immunocytes are taken from the mammaliananimals and supplied for cell fusion, with especially preferredimmunocytes being splenic cells.

[0035] The parent cells used for fusion with the above-mentionedimmunocytes may be myeloma cells from mammalian animals, and a number ofalready publicly known cell strains may be suitably used, including P3(P3x63Ag8.653) (J. Immunol. 123:1548, 1978), p3-U1 (Current Topics inMicrobiology and Immunology 81:1-7, 1978), NS-1 (Eur. J. Immunol.6:511-519, 1976), MPC-11 (Cell, 8:405-415, 1976), SP2/0 (Nature,276:269-270, 1978), Of (J. Immunol. Meth. 35:1-21, 1980), S194 (J. Exp.Med. 148:313-323, 1978), R210 (Nature, 277:131-133, 1979). The cellfusion of the immunocytes with the myeloma cells may be based on apublicly known method, for example the method of Milstein et al.(Milstein et al., Methods Enzymol. 73:3-46, 1981).

[0036] More specifically, the above-mentioned cell fusion is carried outin a conventional nutrient culture in the presence of a cell fusionpromoter. The fusion promoter used may be, for example, polyethyleneglycol (PEG) or Sendai virus (HVJ), and if desired an aid such asdimethylsulfoxide may also be added to increase the fusion efficiency.

[0037] The proportions of the immunocytes and myeloma cells used arepreferably a 1- to 10-fold amount of immunocytes with respect to themyeloma cells. The culturing medium used for the cell fusion may be, forexample, RPMI1640 culture medium or MEM culture medium which aresuitable for growth of myeloma cell strains, or other common culturingmedia used for such cell culturing, and supplementary serum solutionssuch as fetal calf serum (FCS) may also be used therewith.

[0038] The cell fusion is carried out by thoroughly mixing theprescribed amounts of the immunocytes and the myeloma cells in theculture medium described above, adding a PEG solution preheated to about37° C., for example with PEG having an average molecular weight of about1000 to 6000, to the culture medium usually at a concentration of 30 to60% (w/v), and then mixing to form the desired fused cells (hybridomas).Next, the procedure of gradual addition of a suitable culture medium andcentrifugation to remove the supernatant is repeated, to accomplishremoval of the cell fusing agent, etc. which is unfavorable for growthof the hybridomas.

[0039] Suitable hybridomas are selected by culturing in a normalselective culture medium, such as HAT culture medium (containinghypoxanthine, aminopterin and thymine). The culturing in the HAT culturemedium is continued for a given time, usually a few days to a few weeks,sufficient for death of the cells other than the hybridomas (non-fusedcells). Next, normal limited dilution is carried out, and the hybridomasproducing the desired antibodies are subjected to masking andmonocloning.

[0040] The monoclonal antibody-producing hybridomas prepared in thismanner may be subcultured in a common culture solution and they may alsobe placed in liquid nitrogen for long-term storage.

[0041] In order to acquire the monoclonal antibodies from thehybridomas, the hybridomas are cultured according to a conventionalmethod after which the culture supernatant is recovered, or else amethod is used whereby the hybridomas are injected to a compatiblemammalian animal, grown, and the ascites fluid is obtained. The formermethod is suited for obtaining high purity antibodies, while the lattermethod is suited for mass production of the antibodies.

[0042] The monoclonal antibodies obtained by these methods may then bepurified to a high degree using conventional purification means, such assalting-out, gel filtration, affinity chromatography or the like.

[0043] The monoclonal antibodies prepared in this manner may then bechecked for high sensitivity and high purity recognition of the antigenby common immunological means such as radioimmunoassay (RIA),enzyme-linked immunoassay, (EIA, ELISA), the fluorescent antibodytechnique (immunofluorescence analysis), etc.

[0044] The monoclonal antibodies used according to the invention are notlimited to monoclonal antibodies produced by hybridomas, and they may beones which have been artificially modified for the purpose of loweringthe heteroantigenicity against humans. For example, a chimeric antibodymay be used which consists of the variable region of a monoclonalantibody of a mammalian animal other than human, such as a mouse, andthe constant region of a human antibody, and such a chimeric antibodymay be produced by a known chimeric antibody-producing method,particularly a gene recombination technique.

[0045] Reshaped human antibodies may also be used according to theinvention. These are prepared by using the complementary determinantregion of a mouse or other non-human mammalian animal antibody toreplace the complementary determinant region of a human antibody, andconventional gene recombination methods therefor are well-known. One ofthe known methods may be used to obtain a reshaped human antibody whichis useful according to the invention. A preferred example of such areshaped human antibody is hPM-1 (see Intl. Unexamined PatentApplication No. WO92-19759).

[0046] When necessary, amino acids of the framework (FR) region of thevariable region of an antibody may be substituted so that thecomplementary determinant region of the reshaped human antibody forms asuitable antibody binding site (Sato et al., Cancer Res. 53:851-856,1993). In addition, the object stated above may also be achieved byconstructing a gene coding for an antibody fragment which binds to theantigen to inhibit IL-6 activity, such as Fab or Fv, or a single chainFv (scFv) wherein the Fv of the H and L chains are attached via anappropriate linker, and using it for expression in appropriate hostcells (see, for example, Bird et al., TIBTECH, 9:132-137, 1991; Hustonet al., Proc. Natl. Acad. Sci. USA, 85:5879-5883, 1988).

[0047] Modified IL-6 used according to the invention may be the onedisclosed by Brakenhoff et al, J. Biol. Chem. 269:86-93, 1994 or Savinoet al., EMBO J. 13:1357-1367, 1994.

[0048] The modified IL-6 used may be obtained by introducing a mutationsuch as a substitution, deletion or insertion into the IL-6 amino acidsequence to maintain the binding activity with IL-6R while eliminatingthe IL-6 signal transfer function. The IL-6 source may be from anyanimal species so long as it has the aforementioned properties, but interms of antigenicity, a human derived one is preferably used.

[0049] Specifically, the secondary structure of the IL-6 amino acidsequence may be predicted using a publicly known molecular modelingprogram such as WHATIF (Vriend et al., J. Mol. Graphics, 8:52-56, 1990),whereby the influence of mutated amino-acid residues on the entirestructure may also be evaluated. After determining appropriate mutatedamino acid residues, a vector containing the nucleotide sequence codingfor the human IL-6 gene is used as a template for introduction of themutation by the conventionally employed PCR (polymerase chain reaction)method, to obtain a gene coding for the modified IL-6. This is thenincorporated into a suitable expression vector if necessary andexpressed in E. coli cells or mammalian cells, and then used eitherwhile in the culture supernatant or after isolation and purification byconventional methods, to evaluate the binding activity for IL-6R and theneutralized IL-6 signal transfer activity.

[0050] An IL-6 partial peptide or IL-6R partial peptide used accordingto the present invention may have any sequence so long as it binds toIL-6R or IL-6, respectively, and has no IL-6 activity transfer function.IL-6 partial peptides and IL-6R partial peptides are described in U.S.Patent Publication No. U.S. Pat. No. 5,210,075. An IL-6 antisenseoligonucleotide is described in Japanese Patent Application No.5-300338.

[0051] A pharmaceutical composition for treatment of chronic rheumatoidarthritis whose effective component is an IL-6 antagonist according tothe invention is effective for treatment of chronic rheumatoid arthritisif it blocks IL-6 signal transduction and suppresses abnormal growth ofsynovial cells induced by IL-6, which are implicated in the disease.Example 1 demonstrates the in vitro growth suppressing effect onrheumatic patient-derived synovial cells. In Example 2, IL-6 receptorantibody was administered to mice arthritic models immunized with typeII collagen, and the relevant data demonstrates (1) suppression of onsetof arthritis on the basis of an arthritis index (FIG. 4), (2)suppression of anti-type II collagen antibody production in the blood ofcollagen-immunized mice (FIG. 5) and (3) suppression of granulationtissue invasion into cartilage and bone (chronic proliferativesynovitis) in the hind paw joints of mice arthritic models administeredIL-6 receptor antibody (FIG. 6).

[0052] In regard to (1) and (2) above, the results confirmed asuppressing effect by IL-6 receptor antibody, especially initially, ononset of arthritis in the mice models. The results of (3) demonstratedthat invasion of granulation tissue into the cartilage and bone tissueis suppressed, and this supports the results obtained in Example 1 (invitro inhibition of synovial cell growth).

[0053] The experimental results of (1) and (2) indicate that thepharmaceutical composition for treatment of chronic rheumatoid arthritisof the present invention has an excellent initial effect on rheumatoidarthritis.

[0054] The pharmaceutical composition for treatment of chronicrheumatoid arthritis of the invention is preferably administeredparenterally, for example by intravenous, intramuscular, intraperitonealor subcutaneous injection, either systemically or locally. Also, it maybe in the form of a medical formulation kit together with at least onetype of medical carrier or diluent.

[0055] The dosage of the pharmaceutical composition for treatment ofchronic rheumatoid arthritis of the invention when administered tohumans will differ depending on pathological condition and age of thepatient, and the mode of administration, and thus suitable andappropriate doses must be selected. As an example, a maximum of 4divided doses in the range of about 1 to 1000 mg/patient may beselected. However, the pharmaceutical composition for treatment ofrheumatoid arthritis of the invention is not limited to these dosages.

[0056] The pharmaceutical composition for treatment of rheumatoidarthritis of the invention may be formulated according to conventionalmethods. For example, an injection formulation is prepared by dissolvingthe purified IL-6 antagonist in a solvent such as physiological salineor a buffer solution and then adding an adsorption inhibitor such asTween 80, gelatin, human serum albumin (HSA) or the like, and themixture may be lyophilized prior to use for solution reconstitution. Theexcipient used for lyophilization may be a sugar alcohol such asmannitol or glucose, or a saccharide.

EXAMPLES

[0057] The present invention will now be explained in more detail by wayof the following examples, reference examples and experimental examples,with the understanding that the invention is in no way restrictedthereto.

Reference Example 1. Preparation of human soluble IL-6 receptor

[0058] Soluble IL-6R was prepared (Yasukawa et al., J. Biochem.108:673-676, 1990) by the PCR (polymerase chain reaction) method usingplasmid pBSF2R.236 containing cDNA coding for human IL-6 receptor(IL-6R) obtained according to the method of Yamasaki et al. (Science,241:825-828, 1988).

[0059] The aforementioned plasmid pBSF2R.236 was digested withrestriction enzyme SphI to obtain an IL-6R cDNA fragment which was theninserted into mp18 (Amersham Co.). The synthetic oligoprimerATATTCTCTAGAGAGATTCT designed for introduction of a stop codon in IL-6RcDNA was used to introduce a mutation in the IL-6R cDNA by the PCRmethod using an Invitro Mutagenesis System (Amersham Co.). Thisprocedure resulted in introduction of a stop codon at the position ofamino acid 345 to obtain cDNA coding for soluble IL-6R (sIL-6R).

[0060] In order to express the sIL-6R cDNA in CHO cells, theaforementioned sIL-6R cDNA cut with HindIII-SalI was inserted intoplasmid pECEdhfr (Clauser et al., Cell, 45:721-735, 1986) which had cDNAcoding for dihydrofolate reductase (dhfr) inserted at the restrictionenzyme PvuI cleavage site, to obtain the CHO cell expression plasmidpECEdhfr344.

[0061] A 10 μg of plasmid pECEdhfr344 was used for transfection of thedhfr-CHO cell line DXB-11 (Urland et al., Proc. Natl. Acad. Sci. USA 77,4216-4220, 1980) by the calcium phosphate precipitation method (Chen etal., Mol. Cell. Biol. 7:2745-2751, 1987).

[0062] The transfected CHO cells were cultured for 3 weeks in anucleoside-free αMEM selective culture medium containing 1 mM glutamine,10% dialyzed Fetal Calf Serum (FCS), 100 U/ml penicillin and 100 μg/mlstreptomycin. The selected CHO cells were screened by the limitingdilution method, and a single monoclonal CHO cell line was obtained. TheCHO cell clone was amplified in 20 nM to 200 nM concentrationmethotrexate (MTX), to obtain the human sIL-6R-producing CHO cell line5E27.

[0063] The CHO cell line 5E27 was cultured in Iscove's modifiedDulbecco's medium (IMDM, product of Gibco Co.) containing 5% FCS, theculture supernatant was recovered, and the sIL-6R concentration in theculture supernatant was measured by the ELISA (Enzyme-LinkedImmunosorbent Assay) method according to the common procedure.

Reference Example 2. Preparation of human IL-6 antibody

[0064] Human IL-6 antibody was prepared according to the method ofMatsuda et al. (Eur. J. Immunol. 18:951-956, 1988).

[0065] BALB/c mice were immunized with 10 μg of recombinant IL-6 (Hiranoet al., Immunol. Lett., 17:41, 1988) together with Freund's completeadjuvant, and this was continued once a week until anti-IL-6 antibodieswere detected in the blood serum.

[0066] Immunocytes were extracted from the local lymph nodes, andpolyethylene glycol 1500 was used for fusion with the myeloma cell lineP3U1. Hybridomas were selected according to the method of Oi et al.(Selective Methods in Cellular Immunology, W. H. Freeman and Co., SanFrancisco, 351, 1980) using HAT culture medium, and a human IL-6antibody-producing hybridoma line was established. The human IL-6antibody-producing hybridoma was subjected to IL-6 binding assay in thefollowing manner.

[0067] Specifically, a soft polyvinyl 96-well microplate (product ofDynatech Laboratories, Inc., Alexandria, Va.) was coated overnight with100 μl of goat anti-mouse Ig antibody (10 μl/ml, product of CooperBiomedical, Inc., Malvern, Pa.) in a 0.1M carbonate-hydrogen carbonatebuffer solution (pH 9.6) at 4° C. The plate was then treated for 2 hoursat room temperature with PBS containing 100 μl of 1% bovine serumalbumin (BSA). After washing with PBS, 100 μl of hybridoma culturesupernatant was added to each well, and incubation was conductedovernight at 4° C.

[0068] The plates were then washed and ¹²⁵I-labelled recombinant IL-6was added to each well to 2000 cpm/0.5 ng/well, and after washing, theradioactivity of each well was measured with a gamma counter (BeckmanGamma 9000, Beckman Instruments, Fullerton, Calif.). Of 216 hybridomaclones, 32 hybridoma clones were positive for the IL-6 binding assay.Among these clones there was finally obtained the stable cloneMH166.BSF2. The IL-6 antibody MH166 produced by this hybridoma has anIgGIK subtype.

[0069] The IL-6-dependent mouse hybridoma cell line MH60.BSF2 (Matsudaet al., Eur. J. Immunol. 18:951-956, 1988) was then used to determinethe neutralizing activity of MH166 antibody on growth of the hybridoma.MH60.BSF2 cells were dispensed at an amount of 1×10⁴/200 μl/well, asample containing MH166 antibody was added thereto, culture wasperformed for 48 hours, and 15.1 Ci/mmol of ³H-thymidine (New EnglandNuclear, Boston Mass.) was added, after which culture was continued for6 hours.

[0070] The cells were placed on glass filter paper and treated with anautomatic harvester (Labo Mash Science Co., Tokyo, Japan). Rabbitanti-IL-6 antibody was used as a control. As a result, MH166 antibodyinhibited uptake of ³H-thymidine by the MH60.BSF2 cells in adose-dependent manner. This demonstrated that MH166 antibody neutralizesIL-6 activity.

Reference Example 3. Preparation of human IL-6 receptor antibody

[0071] Anti-IL-6R antibody MT18 constructed by the method of Hirata etal. (J. Immunol., 143:2900-2906, 1989) was bound to Sepharose 4B(product of Pharmacia Fine Chemicals, Piscataway, N.J.) activated withCNBr, according to the accompanying instructions, and the bound complexwas used to purify IL-6R (Yamasaki et al., Science 241:825-828, 1988).

[0072] The human myeloma cell line U266 was solubilized with 1 mMp-paraaminophenylmethane sulfonylfluoride hydrochloride (product of WakoChemicals) containing 1% digitonin (product of Wako Chemicals), 10 mmtriethanolamine (pH 7.8) and 0.15M NaCl (digitonin buffer solution), andmixed with MT18 antibody bound to Sepharose 4B beads. The beads werethen washed 6 times with digitonin buffer solution to obtain partiallypurified IL-6R for immunization.

[0073] BALB/c mice were immunized 4 times every 10 days with thepartially purified IL-6R obtained from 3×10⁹ U266 cells, and thenhybridomas were prepared by conventional methods. The culturesupernatants of the hybridomas from the growth-positive wells wereexamined for IL-6 binding activity by the following method. Afterlabelling 5×10⁷ U266 cells with ³⁵S-methionine (2.5 mCi) they weresolubilized with the aforementioned digitonin buffer solution. Thesolubilized U266 cells were mixed with a 0.04 ml of MT18 antibody boundto Sepharose 4B beads, and after washing 6 times with digitonin buffersolution, the ³⁵S-methionine-labelled IL-6R was washed off with 0.25 mlof digitonin buffer solution (pH 3.4) and neutralized with 0.025 ml of1M Tris (pH 7.4).

[0074] A 0.05 ml of the hybridoma culture supernatant was mixed with0.01 ml of Protein G Sepharose (product of Pharmacia). After washing,the Sepharose was incubated with 0.005 ml of the ³⁵S-labelled IL-6Rsolution prepared earlier. The immunoprecipitated substance was analyzedby SDS-PAGE, and the hybridoma culture supernatants reacting with IL-6Rwere examined. As a result, a reaction-positive hybridoma clone PM-1 wasestablished. The IL-6R antibody PM-1 produced by hybridoma PM-1 has anIgGlK subtype.

[0075] The inhibiting activity of the antibody produced by hybridomaPM-1 against binding of IL-6 to human IL-6R was investigated using thehuman myeloma cell line U266. Human recombinant IL-6 was prepared withE. coli (Hirano et al., Immunol. Lett., 17:41, 1988) and ¹²⁵I-labelledwith Bolton-Hunter reagent (New England Nuclear, Boston, Mass.) (Taga etal., J. Exp. Med. 166:967, 1987).

[0076] 4×10⁵ U266 cells were cultured at room temperature in thepresence of a 100-fold excess of non-labelled IL-6 for one hour,together with 70% (v/v) of hybridoma PM-1 culture supernatant and 14000cpm of ¹²⁵I-labelled IL-6. A 70 μl sample was overlaid onto 300 μl ofFCS placed in a 400 μl microfuge polyethylene tube, and aftercentrifugation the radioactivity on the cells was measured.

[0077] As a result it was demonstrated that the antibodies produced byhybridoma PM-1 inhibited binding of IL-6 to IL-6R.

Reference Example 4. Preparation of mouse IL-6 receptor antibody

[0078] Monoclonal antibodies against mouse IL-6 receptor were preparedby the method described in Japanese Patent Application No. 6-134617.

[0079] Following the method of Saito et al. (J. Immunol., 147, 168-173,1993), CHO cells producing mouse soluble IL-6 receptor were cultured inIMDM medium containing 10% FCS, and the mouse soluble IL-6 receptor waspurified from the culture supernatant using the mouse soluble IL-6receptor antibody RS12 (see ibid. Saito et al.) and an affinity columnimmobilizing Affigel 10 gel (Biorad).

[0080] A 50 μg of the obtained mouse soluble IL-6 receptor was mixedwith Freund's complete adjuvant and intraperitoneally injected intowistar rats (Nihon Charles River Co.). Booster immunizations were givenwith Freund's incomplete adjuvant after 2 weeks. On the 45th day therats were butchered, and about 2×10⁸ splenic cells thereof were used forcell fusion with 1×10⁷ mouse P3U1 myeloma cells by a conventional methodutilizing 50% PEG1500 (Berlinger Mannheim), after which the hybridomaswere screened with HAT medium.

[0081] After adding the hybridoma culture supernatants to an immunoplatecoated with rabbit anti-rat IgG antibody (Cappel Co.), mouse solubleIL-6 receptor was reacted therewith and the hybridomas producingantibodies against mouse soluble IL-6 receptor were screened by theELISA method using rabbit anti-mouse IL-6 receptor antibody and alkaliphosphatase-labelled sheep anti-rabbit IgG. The hybridoma clones inwhich antibody production was confirmed were subjected to subscreeningtwice to obtain a single hybridoma clone. This clone was named MR16-1.

[0082] The neutralizing activity of the antibody produced by thishybridoma against mouse IL-6 signal transduction was investigated byincorporation of ³H-thymidine using MH60.BSF2 cells (Matsuda et al., J.Immunol. 18, 951-956, 1988), MH60.BSF2 cells were added to a 96-wellplate to 1×10⁴ cells/200 μl/well, and then mouse IL-6 (10 pg/ml) andMR16-I antibody or RS12 antibody were added to 12.3-1000 ng/ml prior toculturing at 37° C., in 5% CO₂ for 44 hours, after which ³H-thymidine (1μCi/well) was added and the uptake after 4 hours was measured. As aresult, MR16-1 antibody was found to inhibit uptake of ³H-thymidine byMH60.BSF2 cells.

Experiment 1. Establishment of chronic rheumatoid arthritis-derivedsynovial cell line

[0083] (1) Preparation of synovial cells

[0084] Synovial tissue was obtained during surgical operation on thejoint of a chronic rheumatoid arthritis patient. The synovial tissue wasminced with scissors and then subjected to enzymatic dissociation byincubation for one hour at 37° C. with 5 mg/ml of TYPE I collagenase(product of Sigma Chemical Co.) and 0.15 mg/ml of bovine pancreaticDNase (product of Sigma Chemical Co.) in IMDM (Iscove's modifiedDulbecco's medium), and passed through a mesh to obtain singule cells.These obtained cells were then cultured overnight in a culture flaskusing IMDM containing 5% FCS, after which the non-adherent cells wereremoved to obtain the synovial cells. The synovial cells were passaged 3to 6 times and used for the following experiment.

[0085] (2) IL-6 production by synovial cells

[0086] The synovial cells obtained as described above were suspended inIMDM culture medium containing 5% FCS (product of Hyclone LaboratoriesInc.), 10 U/ml of penicillin G and 100 μg/ml streptomycin to an amountof 3×10³ cells/well, and were then cultured in 96-well microtiter plate(product of Falcon Co.), which human interleukin-1β (IL-1β), human tumornecrosis factor α (TNFα), human platelet-derived growth factor (PDGF)ABand human basic fibroblast growth factor (bFGF) were added toconcentrations of 0.01 or 0.1, 0.1 or 1, 1 or 10 and 1 or 10 ng/ml,respectively, and upon culturing at 37° C. for 72 hours the culturesupernatants were collected.

[0087] A 100 μl of anti-human IL-6 antibody MH166 (1 μg/ml) was added toa 96-well ELISA plate (Immunoplate: product of Nunc Co.) and incubatedat 4° C. for 24 hours. Each well was subsequently washed with PBScontaining 0.05% Tween 20, and blocked at 4° C. overnight with PBScontaining 1% BSA. The culture supernatants obtained previously werethen diluted with PBS containing 1% BSA, added to the wells, and thenincubated at room temperature for 2 hours. After washing with PBScontaining 0.05% Tween20, 2.5 μg/ml of rabbit polyclonal anti-human IL-6antibody purified with a 100 μl protein A column (product of Pharmacia)was added.

[0088] After incubating at room temperature for 2 hours, the rabbitpolyclonal anti-IL-6 antibody binding to IL-6 in the culturesupernatants was reacted with alkali phosphatase-bound anti-rabbit IgGantibody (product of Tago Co.). And then 1 mg/ml of Sigma104 alkaliphosphatase substrate (product of Sigma Co.) was added according to theattached instructions and the absorbance at 405-600 nm was measured withan MPR A4 microplate reader (product of Tosoh Co.).

[0089] Calibration curves were prepared for the recombinant IL-6 duringeach assay for conversion of the absorbance OD values to human IL-6concentrations. The results are given in Table 1. TABLE 1 Augmented IL-6production from synovial cell Treatment (ng/ml) IL-6 (ng/ml) Untreated0.096 ± 0.012 IL-1β 0.01 6.743 ± 0.178 0.1 17.707 ± 0.259  TNFα 0.10.575 ± 0.008 1 1.688 ± 0.034 PDGF-AB 1 0.163 ± 0.035 10 0.165 ± 0.016bFGF 1 0.181 ± 0.009 10 0.230 ± 0.019

[0090] The results demonstrated that IL-1β strongly promotes IL-6production by synovial cells.

Example 1

[0091] (1) The synovial cells obtained in Experiment 1 (3×10³/well) weresuspended in IMDM culture medium containing 5% FCS (product of HycloneLaboratories, Inc.), 10 U/ml of penicillin G and 100 μg/ml ofstreptomycin and were then added into a 96-well microtiter plate (#3072,product of Falcon Co.) and cultured for 5 days in the presence ofvarious concentrations of IL-6 or sIL-6 alone, or in the presence ofboth IL-6 and sIL-6R. At 72 hours after starting the culturing,³H-thymidine (product of Amersham International plc) was added to eachwell to 1 μCi/well, and after the culturing was completed theradioactivity in the cells was measured with a scintillation counter.The results are shown in FIG. 1.

[0092] As a result, the ³H-thymidine uptake of the synovial cells waslow with IL-6 or sIL-6R alone, and no growth of synovial cells wasobserved. In contrast, in the presence of at least a 10 ng/mlconcentration of IL-6 and 100 ng/ml concentration of sIL-6R, significantuptake of ^(H)-thymidine was observed compared to the control group.Thus, while virtually no growth effect on synovial cells was exhibitedwith IL-6 alone, in the presence of both IL-6 and sIL-6R a powerfulsynovial cell growth effect was clearly produced.

[0093] (2) Synovial cells (3×10³/well) were cultured in the presence ofa sufficient amount of IL-β to produce IL-6 (0.1 ng/ml), 100 ng/ml ofsIL-6R and 25 μg/ml of IL-6 antibody or 25 μg/ml of IL-6R antibody. At72 hours after the start of culturing, ³H-thymidine was added to eachwell to 1 μCi/well, and after the culture was completed theradioactivity in the cells was measured with a scintillation counter.The results are shown in FIG. 2. Addition of IL-6 antibody or IL-6Rantibody completely suppressed the growth of synovial cells augmented bysIL-6R.

[0094] (3) Synovial cells (3×10³/well) were cultured in the presence of100 ng/ml of IL-6 (product of Genzyme Co.), 100 ng/ml of sIL-6R and 25μg/ml of IL-6 antibody or IL-6R antibody, which were obtained in theabove-mentioned Reference Examples. At 72 hours after the start ofculture, ³H-thymidine was added to each well to 1 μCi/well, and afterthe culture was completed, the radioactivity in the cells was measuredwith a scintillation counter. The results are shown in FIG. 3. Additionof IL-6 antibody or IL-6R antibody completely suppressed the growth ofsynovial cells augmented by sIL-6R.

Example 2

[0095] The suppressing effect of IL-6 receptor antibody on onset ofarthritis was investigated using a mouse arthritis model.

[0096] A bovine type II collagen solution (Collagen Technology ResearchGroup) (4 mg/ml) dissolved in a 0.1N aqueous acetic acid solution andcomplete adjuvant H37Ra (DIFCO) were mixed in equivalent amounts, toprepare an adjuvant. A 100 μl of the adjuvant was subcutaneouslyinjected at the base of tail of 8- to 9-week-old female DBA/1J mice(Charles River Japan). An additional 100 μl was injected 20 days laterunder the dorsal skin to induce arthritis.

[0097] Mouse IL-6 receptor antibody MR16-1 was intravenouslyadministered at 2 mg per mouse upon first collagen sensitization, andeach mouse was subcutaneously injected with an additional 0.5 mg (n=5)each week thereafter for 7 weeks. As a control, anti-DNP antibody KH-5(Chugai Seiyaku) of the same isotype was used (n=5).

[0098] The severity of arthritis was evaluated based on an arthritisindex. The evaluation was based on a 4 point scale for each limb, for atotal of 16 points per individual. The evaluation standard was asfollows.

[0099] 0.5: Erythema observed at one site of joint.

[0100] 1: Erythema observed at two sites of joint, or redness but noswelling of dorsa.

[0101] 2: Moderate swelling observed.

[0102] 3: Severe swelling of pedal dorsa, but not reaching all of thedigits.

[0103] 4: Severe swelling of pedal dorsa and digits.

[0104] The results are shown in FIG. 4. Onset of arthritis from earlystage arthritis was clearly suppressed in the IL-6 receptorantibody-administered group, compared to the controlantibody-administered group.

[0105] On the other hand, the results of measurement of the anti-type IIcollage antibody titer in the mouse blood showed a significant reductionfrom early stage arthritis in the IL-6 receptor antibody-administeredgroup compared to the control antibody-administered group (FIG. 5).

[0106] The mice were sacrificed on the 35th day after collagenimmunization, and the hind legs were fixed with 20% formalin. They werethen subjected to demineralization in an EDTA solution (pH 7.6) anddewatering with alcohol. They were subsequently wrapped in paraffin andcut to 2 μm thick sections. The sections were stained with hematoxylinand eosin and observed under 125× magnification (FIG. 6). As a result,invasion of granulation tissue into the cartilage and bone, i.e. chronicproliferative synovitis was suppressed in the IL-6 receptorantibody-administered group compared to the controlantibody-administered group.

[0107] IL-6 is a cytokine which induces differentiation of B cells intoantibody-producing cells. IL-6 also promotes proliferation of synovialcells in the presence of IL-6 receptor. Since in mouse collagenarthritis models, anti-IL-6 receptor antibody significantly suppressedanti-type II collagen antibody titers on the 21st and 35th days aftercollagen sensitization, compared to the control antibody-administeredgroup, it is believed that the antibody production inhibition byanti-IL-6 receptor antibody is one factor responsible for thesuppressing effect on arthritis. Moreover, although no suppression ofantibody production was observed from the 49th day after collagensensitization, the fact that an adequate suppressing effect on onset ofarthritis was exhibited even during this period, and that HE staining oftissue surrounding the tarsal bone showed suppressed invasion ofgranulation tissue into the cartilage and bone of the anti-IL-6 receptorantibody-administered group compared to the control group, the synovialgrowth-suppressing effect is also believed to contribute to thearthritis-inhibiting effect.

INDUSTRIAL APPLICABILITY

[0108] Synovial cells from chronic rheumatoid arthritis patientsproliferate in the presence of both IL-6 and sIL-6R. The fact thatsynovial fluid of chronic rheumatoid arthritis patients contains asufficient amount of IL-6 and sIL-6R to induce growth of synovial cellssuggests that signal transduction by IL-6 is involved in abnormal growthof synovial cells in chronic rheumatoid arthritis.

[0109] It has thus been conclusively demonstrated that a chronicrheumatoid arthritis therapy whose effective component is an IL-6antagonist according to the present invention suppresses growth ofsynovial cells in chronic rheumatoid arthritis patients in the presenceof IL-6 and sIL-6R, and thus has a therapeutic effect against chronicrheumatoid arthritis. Consequently, the IL-6 antagonist of the inventionis useful as a therapeutic agent for chronic rheumatoid arthritis inwhich abnormal growth of synovial cells occurs.

1. A pharmaceutical composition for treatment of chronic rheumatoidarthritis containing an interleukin-6 antagonist as an effectivecomponent.
 2. A pharmaceutical composition for treatment of chronicrheumatoid arthritis according to claim 1 , characterized in that saidinterleukin-6 antagonist suppresses abnormal growth of synovial cellsoccurring with chronic rheumatoid arthritis.
 3. A pharmaceuticalcomposition for treatment of chronic rheumatoid arthritis according toclaim 1 , characterized in that said interleukin-6 antagonist is anantibody against interleukin-6.
 4. A pharmaceutical composition fortreatment of chronic rheumatoid arthritis according to claim 2 ,characterized in that said interleukin-6 is human interleukin-6.
 5. Apharmaceutical composition for treatment of chronic rheumatoid arthritisaccording to claim 1 , characterized in that said interleukin-6antagonist is an antibody against interleukin-6 receptor.
 6. Apharmaceutical composition for treatment of chronic rheumatoid arthritisaccording to claim 2 , characterized in that said interleukin-6 receptoris human interleukin-6 receptor.
 7. A synovial cell growth inhibitorcontaining an interleukin-6 antagonist as an effective component.
 8. Asynovial cell growth inhibitor according to claim 7 , characterized inthat said interleukin-6 antagonist is interleukin-6 antibody orinterleukin-6 receptor antibody.